Identification of small molecule inhibitors of cytokinesis and single cell wound repair.

Department of Zoology, University of Wisconsin-Madison, Madison, WI 53706, USA.

Abstract

Screening of small molecule libraries offers the potential to identify compounds that inhibit specific biological processes and, ultimately, to identify macromolecules that are important players in such processes. To date, however, most screens of small molecule libraries have focused on identification of compounds that inhibit known proteins or particular steps in a given process, and have emphasized automated primary screens. Here we have used "low tech" in vivo primary screens to identify small molecules that inhibit both cytokinesis and single cell wound repair, two complex cellular processes that possess many common features. The "diversity set", an ordered array of 1990 compounds available from the National Cancer Institute, was screened in parallel to identify compounds that inhibit cytokinesis in Dendraster excentricus (sand dollar) embryos and single cell wound repair in Xenopus laevis (frog) oocytes. Two small molecules were thus identified: Sph1 and Sph2. Sph1 reduces Rho activation in wound repair and suppresses formation of the spindle midzone during cytokinesis. Sph2 also reduces Rho activation in wound repair and may inhibit cytokinesis by blocking membrane fusion. The results identify two small molecules of interest for analysis of wound repair and cytokinesis, reveal that these processes are more similar than often realized and reveal the potential power of low tech screens of small molecule libraries for analysis of complex cellular processe.

Schematic showing overview of chemical screen and structure of the two compounds identified by it

A. In the primary screen, 1990 small molecules from the diversity kit were screened in parallel for their ability to inhibit wound healing in Xenopus oocytes or cytokinesis in sand dollar embryos. In the secondary screen, compounds that inhibited both processes were then then subjected to toxicity and ATP production screens. Two small molecules--NSC2805 and NSC292596 were positive in the primary screen and negative in the secondary screen. Numbers indicate hits/total compounds. B. Structures of NSC2805 and NSC292596.

A. Light micrographs from time lapse movies showing first mitosis in sand dollar embryos treated with DMSO (Con; 0.1%), Sph1 (4 μM) or Sph2 (5 μM). Images begin with start of anaphase with asters indicated by arrows. Time in min:sec. B. Light micrographs from time lapse movies showing several mitoses in sand dollar embryos treated with DMSO (Con; 0.1%), Sph1 (3 μM) or Sph2 (8 μM). Arrows at 01:20:00 time point in Sph1 and Sph2 samples show multiple asters in common cytoplasm. Time in hour:min:sec. C. Light micrographs from time lapse movies showing first early mitoses in Xenopus embryos treated with DMSO (Con; 0.1%), Sph1 (50 μM) or Sph2 (50 μM). Arrows in Sph1, 30:00 time point show furrows that subsequently regress. Double arrows in Sph2 53:00 show regions of membrane tearing that flank closing cleavage furrow. Time in min:sec and hour:min:sec.